Compositions and methods for identifying agents which regulate chromosomal stability, gene activation and aging

ABSTRACT

Novel compositions and methods are provided for identifying agents which affect chromosomal stability and aging.

[0001] Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health,Grant Number: GM59785.

FIELD OF THE INVENTION

[0002] Several publications are referenced in this application bynumbers in parentheses in order to more fully describe the state of theart to which this invention pertains. The disclosure of each of thesepublications is incorporated by reference herein.

[0003] The SIR2 (silent information regulator 2) gene family of proteinsis conserved from bacteria to man (1). In yeast, SIR2 is required fortranscriptional silencing (2), and is also involved in suppressing rDNArecombination and controlling life span (3, 4). Very little is knownabout the roles of four other SIR2 family members found in yeast (5),nor about the function of homologs from other species. The Salmonellatyphimurium homolog, CobB, can substitute for CobT as a phosphoribosyltransferase during cobalamin biosynthesis (6). In-vitro, CobB hashistone/protein deacetylase activity (7, 8). Given the widespreadoccurrence and extensive conservation of the SIR2-like proteins,understanding their molecular mechanism is essential for identifying thecellular roles played by these proteins. Unfortunately, discrepanciesamong recent reports (7-11) have added to the general uncertainty as tothe true enzymatic function for this family of proteins. These reportshave suggested that the SIR2 proteins are either histoneADP-ribosyltransferases (9, 11), or histone/protein deacetylases (7, 8),or both (10).

SUMMARY OF THE INVENTION

[0004] In accordance with the present invention, a novel acetyl-ADPribose compound (O-acetyl-ADP ribose) has been identified as the primaryproduct of the histone deacetylase reaction catalyzed by the HTS2protein. The availability of O-acetyl-ADP ribose facilitates productionof immunologically specific antibodies for isolating this molecule. Suchantibodies may be polyclonal or monoclonal. Antibodies immunologicallyspecific for O-acetyl ADP ribose may be used to advantage in competitivebinding assays to identify test compounds having affinity for O-acetylADP ribose. Test compounds so identified are also within the scope ofthe present invention.

[0005] Methods for producing the novel O-acetyl ADP ribose of theinvention as well as analogs thereof are also provided.

[0006] In yet a further aspect, pharmaceutical compositions containingthe novel compounds of the invention are provided. Such pharmaceuticalcompositions should have efficacy as anti-aging and anti-cancer agents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 shows reverse phase HPLC elution of substrate standards(NAD⁺ and AcLys-14H3 peptide) and of potential products (H3 peptide,acetate, ADP ribose, and nicotinamide). Approximately 1.5 nmol ofAcLys-14H3 peptide, 1.0 nmol of H3 peptide, 10 nmol NAD, 15 nmol ADPribose, and 15 nmol of nicotinamide were mixed and subjected to reversephase chromatography. In a separate HPLC run, 1.0 nmol ofNa-[³H]-acetate (100,000 cpm/nmol) was subjected to chromatography.Order of elution: nicotinamide, acetate, ADP-ribose, NAD⁺, H3 peptideand AcLys-14H3 peptide. The elution position of acetate (denoted with anasterisk) was determined using authentic [³H]-acetate and detection byliquid scintillation counting. All others were detected by UV absorptionat 214 nm.

[0008]FIG. 2 shows elution of products from the HST2 NAD-dependentdeacetylation reaction, detected by UV absorbance at 214 nm. Novelproduct is denoted with an asterisk. Conditions: 3.6 μM HST2, 175 μMNAD⁺, 525 μM Lys14 AcH3, 1 mM DTT, 37° C. for 30 minutes prior toquenching with TFA to final concentration of 1%.

[0009]FIG. 3 is a graph showing that the amount of deacetylationcorrelates exactly with the consumption of NAD⁺. This graph displays theprogress curves of deacetylation at fixed, but limiting [NAD⁺]. HST2reaction was allowed to proceed to completion under various limiting[NAD⁺] and the amount of deacetylated H3 peptide was determined.Conditions: 375 nM HST2, 175 μM Lys14 AcH3, 8.75, 17.5, or 35 μm NAD⁺, 1mM DTT, 37° C. for 20 min prior to quenching with TFA to 1%.

[0010]FIG. 4 is a graph showing steady-state rate of nicotinamide anddeacetylated H3 peptide formation at various fixed [NAD⁺] Conditions:375 nM HST2, 175 μM Lys14 AcH3, 8.75 μM to 280 μM NAD⁺, 1 MM DTT, 37° C.for 1 min prior to quenching with TFA to a final concentration of 1%.

[0011] FIGS. 5A-5C are a series of graphs showing that acetate is not aprimary product of HST2-catalyzed histone/protein deacetylation. H3peptide was stoichiometrically acetylated on Lys-14 by the histoneacetyltransferase P/CAF and using [³H]-AcCoA. Monoacetylated[³H]-AcLys-14H3 peptide was then purified by HPLC (FIG. 5A) and utilizedas a substrate in the HST2 deacetylation reactions (FIG. 5B). Uponcomplete consumption of [³H]-AcLys-14H3 by HST2, all of the original[³H] from H3 peptide was converted to a labeled product that eluted muchlater than authentic acetate. Conditions: 375 nM HST2, 175 μM NAD⁺, 5 μM[³H]-Lys14 AcH3, 1 mM DTT, for 1 min at 37° C. prior to quenching withTFA to a final concentration of 1%. FIG. 5C: Single turnover rapidquench-flow analysis. HST2 (10 mM) and NAD⁺ (300 mM) were rapidly mixedwith 2.5 mM [³H]-AcLysl4H3 peptide at 22±3° C., pH 7.5 in a Hi-TechRapid Quench-Flow Device RQF-63. Between 31 msec-8 sec, reactions werequenched with TFA (1%). Quantification of [³H]-AcLys14H3 peptide(diamonds) and the [³H]-acetate-adduct (circles) was accomplished byliquid scintillation counting of these species separated using reversephase HPLC. Data were fitted to a single exponential, yielding rateconstants of 2.0±0.1 s⁻¹ for [³H]-AcLysl4H3 peptide deacetylation and2.3±0.2 s⁻¹ for [³H)-acetate-adduct formation.

[0012]FIGS. 6A and 6B show mass spectral analysis of the novel productgenerated by HST2-catalyzed histone/protein deacetylation. MALDI massspectrometry was used to identify a mass of 602, consistent with theformation of acetyl-ADP ribose (O-acetyl-ADP ribose; FIG. 6A). Forcomparison, authentic ADP ribose yielded a predicted mass of 560 (FIG.6B). With both ADP ribose and acetyl-ADP ribose, masses corresponding tothe association of 1 and 2 sodium ions were also observed.

[0013]FIG. 7 shows a proposed catalytic mechanism for the production ofO-acetyl-ADP ribose. Acetyl-lysine condenses directly with theoxo-carbenium cation, which resulted from the elimination ofnicotinamide from NAD⁺. A hydroxide ion then attacks this intermediateto form a tetravalent intermediate, which can collapse to produce1-O-acetyl-ADP ribose (Compound I, FIG. 7). The 1-O-acetyl-ADP ribosemay then be attacked by the adjacent 2′ OH group (via enzyme-assistedgeneral base catalysis)′ to produce the 2-O-acetyl-ADP ribose (CompoundII, FIG. 7). Once in bulk solution, the 2-O-acetyl-ADP ribose appears tobe in rapid equilibrium with its solution migration adduct3-O-acetyl-ADP ribose (Compound III, FIG. 7). Additional quantitativeHPLC analysis followed by 2-dimentional NMR (heteronuclear multiple bondcorrelation) experiments have revealed the migration and equilibriumamong the O-acetyl ADP ribose adducts depicted in FIG. 7. The enzymaticreaction could occur in either stepwise or concerted fashion. Forclarity, we have drawn the chemical events as stepwise events.

[0014]FIGS. 8A and 8B are graphs which clearly demonstrate that proteinspresent in a human cell extract convert O-acetyl-ADP ribose to adistinct, but as yet unidentified species. These findings implicateO--acetyl-ADP ribose as a second messenger, where additionalproteins/enzymes are capable of terminating the signal through theenzymatic decomposition of O-acetyl-ADP ribose. These data also provideevidence for the existence of proteins/enzymes that bind/utilizeO-acetyl-ADP ribose.

[0015] FIGS. 9A-9C are an elution trace and a pair of micrographs. FIG.9A, HPLC (reversed phase) elution trace of O-acetyl-ADP-riboseenzymatically produced by the NAD-dependent deacetylase HST2. Theproduct was purified using two sequential reversed phase separations.Vertical marks are strip-chart ticks of the fraction collector. The peakwas collected and lyophilized; the product was then resuspended inTFA/acetonitrile/water and re-lyophilized to complete dryness until use.Purified O-acetyl-ADP-ribose was solubilized in PBS and microinjectedinto immature starfish oocytes at final 1 mg/mL. Maturation hormone(1-methyladenine) was then added, and images were taken at 3 (FIG. 9B)and 24 hr (FIG. 9C) post hormone treatment. Control oocytes wereinjected with PBS. By 3 hr, normal/control oocytes display GV breakdown,and by 24 hr (if not fertilized) will undergo programmed cell death(apoptosis) (see controls). The O-acetyl-ADP-ribose injected oocytes didnot undergo GV breakdown or polar body extrusion, nor did they undergoapoptosis. Instead, they remained in a state of stasis.

[0016] FIGS. 10A-C show HST2 micro-injection analysis of oocytedevelopment. (FIG. 10A) The egg on the right was injected with 200 nMfinal HST2, the oocytes were then matured by addition of 10 μM 1-MA, andfertilized 4 hours later. The control egg matured, and fertilized whilethe injected egg failed to mature, and therefore was incompetent forfertilization. 10 of 10 eggs injected in this chamber behaved the sameway. (FIG. 10B) The egg on the right is injected with R45AHST. Theoocytes were matured by addition of 10 μM 1-MA, and fertilized fourhours later. Both eggs matured and were fertilized normally 4 hourslater. 7 of 7 injected eggs in the same chamber behaved the same way.(FIG. 10C) One blastomere of the 2 cell stage embryo on the left wasinjected with 200 nM HST2 WT. The injection was done after completecytokinesis. The injected blastomere divided once, then both daughtersstopped, while the sister which saw no dose, went on to form a normalblastula.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Conflicting reports have suggested that the SIR2 (silentinformation regulator 2) protein family employs NAD⁺ to eitherADP-ribosylate histones (1, 9), or deacetylate histones (7, 8) or both(10). Uncovering the true enzymatic function of SIR2 is critical to thebasic understanding of its cellular function. Therefore, we set out toauthenticate the reaction products and to determine the intrinsiccatalytic mechanism. We provide direct evidence that the efficienthistone/protein deacetylase reaction is tightly coupled to the formationof a novel acetyl-ADP ribose product (O-acetyl-ADP ribose). One moleculeof NAD⁺ and acetyl-lysine are readily catalyzed to one molecule ofdeacetylated lysine, nicotinamide, and o-acetyl-ADP ribose. A uniquereaction mechanism involving the attack of enzyme-bound acetate ordirect attack of acetyl-lysine on an oxo-carbenium ADP riboseintermediate is proposed. It appears that the previously reportedhistone/protein ADP-ribosyltransferase activity is a low efficiency sidereaction that can be explained through the partial uncoupling of theintrinsic deacetylation and acetate transfer to ADP ribose. Alsoprovided herein is data revealing that O-acetyl-ADP ribose plays apivotal role in cell cycle control. This observation provides supportfor the utilization of O-acetyl-ADP ribose and its analogs asanti-cancer agents and anti-aging compounds.

[0018] I. Antibodies Immunologically Specific for O-Acetyl-Ribose

[0019] The present invention also provides antibodies capable ofimmunospecifically binding to O-acetyl-ADP ribose. Polyclonal antibodiesdirected toward O-acetyl-ADP ribose may be prepared according tostandard methods. In a preferred embodiment, monoclonal antibodies areprepared, which react immunospecifically with various epitopes ofO-acetyl-ADP ribose. Monoclonal antibodies may be prepared according togeneral methods of Kohler and Milstein, following standard protocols.Polyclonal or monoclonal antibodies that immunospecifically interactwith O-acetyl-ribose can be utilized for identifying and purifying thecompound. For example, antibodies may be utilized for affinityseparation of proteins with which they immunospecifically interact.Antibodies may also be used to identify cells containing O-acetyl-ADPribose. O-acetyl-ribose-specific antibodies may also be used toimmunoprecipitate proteins which bind O-acetyl-ribose from a samplecontaining a mixture of proteins and other biological molecules. Otheruses of anti-O-acetyl-ribose antibodies are described below. Antibodiesaccording to the present invention may be modified in a number of ways.Indeed the term “antibody” should be construed as covering any bindingsubstance having a binding domain with the required specificity. Thus,the invention covers antibody fragments, derivatives, functionalequivalents and homologues of antibodies, including synthetic moleculesand molecules whose shape mimics that of an antibody enabling it to bindan antigen or epitope.

[0020] Exemplary antibody fragments, capable of binding an antigen orother binding partner, are Fab fragment consisting of the VL, VH, C1 andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

[0021] Humanized antibodies in which CDRs from a non-human source aregrafted onto human framework regions, typically with alteration of someof the framework amino acid residues, to provide antibodies which areless immunogenic than the parent non-human antibodies, are also includedwithin the present invention.

[0022] I. Rational Drug Design

[0023] According to one aspect of the invention, methods of screeningagents to identify suitable drugs for inhibiting or augmenting thehistone/protein deacetylase reaction are provided herein.

[0024] O-acetyl-ADP ribose employed in protein binding assays may eitherbe free in solution, affixed to a solid support or within a cell. Onemethod of screening for compounds which bind 1-O-acetyl-ribose utilizeseukaryotic or prokaryotic host cells which are stably transformed withrecombinant polynucleotides expressing the HST2 or SIR2 gene polypeptideor fragment, preferably in competitive binding assays. Such cells,either in viable or fixed form, can be used for standard binding assays.One may determine, for example, formation of complexes between aO-acetyl-ribose and a protein present in the cell. Alternatively, suchcells may be contacted with test compounds to identify novel testcompounds which bind either the deacetylase enzyme or the O-acetylribose reaction product. Such assays provide the means to assess thedegree to which the formation of a complex between the deacetylaseenzyme or O-acetyl-ribose and a known ligand is interfered with by theagent being tested.

[0025] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity toO-acetyl-ribose and is described in detail in Geysen, PCT publishedapplication WO 84/03564, published on Sep. 13, 1984. Briefly stated,large numbers of different, small peptide test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thepeptide test compounds are reacted with O-acetyl-ribose and washed.Bound O-acetyl-ribose is then detected by methods well known in the art.

[0026] Purified O-acetyl-ribose can also be coated directly onto platesfor use in the aforementioned screening techniques. This invention alsocontemplates the use of competitive drug screening assays in whichneutralizing antibodies capable of specifically binding theO-acetyl-ribose compete with a test compound for binding to theO-acetyl-ribose. In this manner, the antibodies can be used to detectthe presence of molecules which share one or more antigenic determinantsof the O-acetyl-ribose compound.

[0027] The goal of rational drug design is to produce structural analogsof biologically active polypeptides of interest or of small moleculeswith which they interact (e.g., agonists, antagonists, inhibitors) inorder to fashion drugs which are, for example, more active or stableforms of the compound of the invention, or which, e.g., enhance orinterfere with the function of a O-acetyl ribose in vivo. See, e.g.,Hodgson, (1991) Bio/Technology 9:19-21. In one approach, one firstdetermines the three-dimensional structure of a small molecule ofinterest (e.g., O-acetyl-ribose) or, for example, of theO-acetyl-ribose-deacetylase complex, by x-ray crystallography, bynuclear magnetic resonance, by computer modeling or most typically, by acombination of approaches. Less often, useful information regarding thestructure of a compound may be gained by modeling based on the structureof similar compounds. An example of rational drug design is thedevelopment of HIV protease inhibitors.

[0028] It is also possible to isolate a target-specific antibody,selected by a functional assay, and then to solve its crystal structure.In principle, this approach yields a pharmacore upon which subsequentdrug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original molecule. The anti-id couldthen be used to identify and isolate small molecules from banks ofchemically or biologically produced banks of molecules. Selectedmolecules would then act as the pharmacore.

[0029] Thus, one may design drugs which have, e.g., improvedO-acetyl-ribose activity or stability or which act as inhibitors,agonists, antagonists, etc. of the reaction in which O-acetyl-ribose isformed. By virtue of the availability of isolated O-acetyl-ribose,sufficient amounts of the compound may be made available to perform suchanalytical studies as x-ray crystallography. In addition, the knowledgeof the O-acetyl-ribose structure provided herein will guide thoseemploying computer modeling techniques in place of, or in addition tox-ray crystallography.

[0030] III Therapeutics

[0031] Agents identified by the above described methods of the inventioncan be formulated in pharmaceutical compositions for the treatment ofcancer and aging. Changes in histone acetylation levels play a centralrole in the regulation of neoplasia, tumor suppression, cell cyclecontrol, hormone response, chromosome stability and senescence.Reversible histone acetylation controls gene transcription. The properlevel of histone acetylation is regulated by histone acetyltransferaseactivity and by histone deacetylase activity. Recent results on genespecific effects have provided a rationale basis for the development ofhistone deacetylase inhibitors as antitumor agents. Furthermore, manytumor suppressor genes are known to be inactivated by promoter DNAhypermethylation, a process which is closely linked to histonedeacetylation and chromatin structure. DNA methylation and histonedeacetylation act synergistically in silencing genes involved in cancer.Also, many acute leukemias are caused by fusion proteins that have beencreated by chromosomal translocations involving histoneacetyltransferases.

[0032] The SIR2 NAD-dependent histone deacetylase also appears to play arole in aging (18). Recently, the observation that caloric restrictionleading to increased life-span appears to be linked through an NAD⁺ andSIR2-dependent pathway (18), raising the possibility that O-acetyl-ADPribose or its direct metabolites may play a role in this phenomenon.This finding indicates that O-acetyl-ADP ribose or its analogs mayaffect the anti-aging process when taken as a therapeutic or as adietary supplement.

[0033] These compositions may comprise, in addition to one of the abovesubstances, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material may depend on the route of administration, e.g. oral,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraperitoneal routes.

[0034] Whether it is a polypeptide, antibody, peptide, nucleic acidmolecule, small molecule or other pharmaceutically useful compoundaccording to the present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual.

[0035] The following materials and methods are provided to facilitatethe practice of the present invention.

[0036] Materials. [³H]Acetyl-CoA (1.88 Ci/mmol) and Na-[³H]-acetate(100,000 cpm/nmol) was from NEN Life Sciences Products. Nicotinamide,ADP-ribose, and nicotinamide adenine dinucleotide (NAD) were purchasedfrom Sigma Chemical Company. Histone H3 peptide, ARTKQTARKSTGGKAPPKQLCand the Lysine 14 acetylated H3 peptide (AcLys-14), corresponding to the20 amino-terminal residues of human histone H3 was synthesized by theProtein Chemistry Core Lab at Baylor College of Medicine. Histidinetagged full length HST2 was recombinantly expressed and purified fromEscherichia coli BL21DE3 bacteria using a T7 polymerase-basedexpression. Harvested cells were lysed by French pressure in 50 mM Tris,pH 8.0, 300 mM NaCl, 1 mM β-mercaptoethanol with protease inhibitors(0.1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, and 5 μg/mLaprotinin). The clarified extract was rocked batchwise withNi-NTA-agarose (Qiagen) (2 mL) for 1 h at 4° C. The Ni-NTA-agarose wasthen applied to a small column and washed with 50 mM Tris, pH 8.0, 300mM NaCl, 1 mM 2-mercaptoethanol. HST2 protein was eluted with a linear0-500 mM imidazole gradient. HST2 eluted at 200 mM imidazole and wasdetermined to be >95% pure by scanning densitometry of commassie-stainedSDS-PAGE. Imidazole was remove by extensive dialysis in the abovementioned Tris buffer. CobB and SIR2 proteins were purified as described(7).

[0037] Catalytic Analysis by HPLC. Standards of AcLys-14H3 peptide andNAD⁺ (HST2 substrates), and potential products of the HST2 reaction, H3peptide, nicotinamide, [³H]-acetate and ADP-ribose were resolved byreverse phase HPLC on a Beckman Ultrasphere column (4.6 mm by 15 cm).Samples were injected in 0.05% TFA/H₂O for 1 min prior to a lineargradient of 0-40% acetonitrile in 1-41 min. The peaks of absorbance at214/260 nm were collected and subjected to mass spectral analysis.[³H]-acetate was detected by liquid scintillation counting. Toauthenticate the reaction products, HST2 (3.6 μM) was mixed withsubstrates AcLys-14 H3 peptide (525 μM) and NAD⁺ (175 μM) and allowed toreact at 37° C. for 5 min prior to quenching with TFA to 1%. Substratesand products were resolved on reverse phase HPLC and subjected to massspectral analysis.

[0038] Typical HST2 steady-state reactions to monitor deacetylation ofAcLys-14H3 peptide or nicotinamide formation were carried out at 37° C.in 50 mM Tris, pH 7.5 buffer. Rates of product formation were determinedat various concentrations of AcLys-14H3 peptide (5-300 μM) and NAD⁺(5-600 μM). During the linear portion of the initial velocity, thereactions were quenched with TFA to 1% and product formation wasmonitored by the HPLC analysis described above. Standard curves weregenerated to quantify H3 peptide and nicotinamide product formation byinjecting known amounts of authentic standards and monitoring thecorresponding peak height at 214 nm. The standard curves were linear inthe range of 0-2 nmol and were reproducible from day to day.

[0039] P/CAF [³H]-monoacetylation of H3 peptide. The histoneacetyltransferase P/CAF was used to monoacetylate H3 peptide on Lysine14 (12). P/CAF (0.175 μM was mixed with [³H]-acetyl CoA (33.3 μM, 1.88Ci/mmol) and H3 peptide (175 μM) for 20 min at 25° C. prior to quenchingwith TFA to 1%. Mono [³H]-acetylated H3 peptide was separated fromunmodified H3 by HPLC analysis as described above.

[0040] MALDI/ESI Mass Spectrometry Analyses. MALDI mass spectrometry wasperformed (Oregon State University, Environmental Health SciencesCenter) on a custom-built reflector time-of-flight mass spectrometerequipped with a two-stage delayed extraction source; Approximately 1 μLof sample solution was mixed with 2 μL 2,4,6-THAP (2,4,6-trihydroxyacetophenone; 10 mg/ml in 70:30 water/acetonitrile, plus 50 mg/mLdiammonium citrate in water); a 1.0 μL droplet of this analyte/matrixsolution was deposited onto a matrix precrystallised sample probe andallowed to dry in air. Mass spectra were produced by irradiating thesample with a (355 nm) Nd:YAG laser (Spectra Physics) and operating theion source at 23 kV with a 150 ns/1.0 kV delay. Every mass spectrum wasrecorded as the sum of 20 consecutive spectra, each produced by a singlepulse of photons. Ions from an added standard were used for masscalibration. ESI mass spectrometry was also carried out at EmoryUniversity School of Medicine Microchemical Facility. Precursor ion(phosphate anion) scanning procedure was performed as described (13).Chemical data base searches (Available Chemical Directory Data Base, MDLInformation Incorporated; cas-online, American Chemical Society;SciFinder Scholar, American Chemical Society) revealed that O-acetyl ADPribose is a novel compound.

[0041] Rapid-Reaction Kinetic Analysis. For the quench-flow analysisunder single turnover conditions, HST2 (10 μM) and NAD⁺ (300 μM), wererapidly mixed with 2.5 μM [³]-AcLys14H3 peptide at 22±3° C., pH 7.5 in aHi-Tech Rapid Quench-Flow Device RQF-63. After various reactions timesbetween 31 msec-8 sec, the reactions were quenched with TFA to a finalconcentration of 1%. The amount of [³H]-AcLys14H3 peptide and(³H]-O-acetyl ADP ribose was determined by liquid scintillation countingof these species separated on reverse phase HPLC.

[0042] Methods for monitoring O-acetyl-ADP ribose utilization by enzymesfrom human Hela cell extracts. HeLa Cells were grown at 37° C. inDulbecco's Modified Eagles Medium containing D-glucose 1 g/L,L-glutamine 1 g/L, pyridoxine hydrochloride 1 g/L, sodium pyruvate 110mg/L, 10% fetal bovine serum (FBS), penicillin at 1000 units/mL, andstreptomycin at 1 mg/mL (Gibco BRL) (growth medium). When cells hadreached 80-90% confluency, they were rinsed once with PBS, lysed in100-200 μL of ice-cold, 20 mM Tris, pH 7.2, 137 mM NaCl, 10% glycerol,1% Nonidet P-40, 100 μM phenylmethylsulfonyl fluoride (PMSF), 20 μg/mLleupeptin, and 20 μg/mL of aprotinin. Cells were scraped into eppendorftubes, sonicated for 10s, and centrifuged at 16,000×g for 10 min toremove cell debris. Protein concentrations of supernatants weredetermined by the method of Bradford. Lysates (40 μL) of the cells orbuffer controls were then incubated with radiolabeled 1-O-acetyl-ADPribose (about 0.5 nmol) (total volume of reaction was 100 μL) for theindicated times prior to analysis by reverse phase HPLC. The HPLCanalysis was carried out as described previously.

[0043] Collection of Gametes and Microinjection

[0044]Asterina miniata gametes were collected and microinjections wereperformed as described previously (Carroll et al., 1999). Briefly,quantitative microinjection of Asterina miniata oocytes was performed at16° C., using mercury-filled micropipets (Hiramoto, 1962; Kiehart, 1982;and see the information available at http://egg/uchc.edu/injection),allowing injection of precisely calibrated picoliter volumes into theoocytes and blastomeres. Injection volumes were 3% of the total cellularvolume (for oocytes, 93 pL and for blastomeres, 47 pL). Buffer alone,purified O-acetylated ADP ribose or recombinant protein was injectedinto immature oocytes or one blastomere of a two cell stage embryo to afinal concentration in the cytoplasm as indicated in the figure legendsand Tables. 1-methyl adenine was from Sigma and was used at 10 μM.

[0045] Preparation of Oocyte Extracts

[0046] Oocyte suspensions were centrifuged (280×g) briefly and theseawater was removed. Oocytes were resuspended in lysis buffer (PBS, pH7.4, 15 mM disodium-EGTA, 1% Triton x-100, 0.5 mM sodium vanadate, 1 mMsodium fluoride, 10 μM each of aprotonin, leupeptin, and benzamadine, 50μM PMSP) and lysed by passage through a 27.5 gauge needle on ice. Thesample was centrifuged at 18,000×g at 4° C. for 20 min and the solublefraction was collected and kept on ice. Protein concentration wasdetermined using the Pierce BCA assay using BSA as a standard. Aliquotswere snap frozen in liquid nitrogen and stored at −80° C.

[0047] The following examples are provided to illustrate variousembodiments of the invention. They are not intended to limit theinvention in any way.

EXAMPLE I

[0048] Due to the high yield and activity of recombinant enzyme, theyeast SIR2 homolog HST2 was utilized as the prototypical SIR2 member forthe extensive study described here. Where noted, yeast SIR2 and theEscherichia coli homolog, CobB, were employed to demonstrate theconservation in catalytic function among the family. Although therequirement for NAD⁺ has been demonstrated, surprisingly, the trueidentity and stoichiometry of the products has not been investigated.The extent of coupling between NAD⁺ consumption and histonedeacetylation was examined initially using the monoacetylated histone H3peptide ARTKQTARKSTGG(AcK)APRKQL (corresponding to the first 20amino-terminal residues in histone H3), subsequently referred to asAcLys-14H3 peptide. To unambiguously identify the authentic products ofthe NAD-dependent deacetylation reaction, HST2 catalyzed products wereresolved and quantified by reverse phase HPLC, and verified by massspectrometry. First, standards of substrates (NAD⁺ and AcLys-14H3peptide) and of the potential products (H3 peptide, acetate, ADP ribose,and nicotinamide) were separated on reverse phase HPLC (FIG. 1). Thecomponents eluted in the order: nicotinamide, acetate, ADP-ribose, NAD⁺,H3 peptide and AcLys-14H3 peptide. The elution position of acetate wasdetermined using [³H]-acetate and detection by liquid scintillationcounting, whereas all others were detected by UV absorption at 214 nm or260 nm (FIG. 1).

[0049] HST2 deacetylation reactions in the absence of NAD+produced nodetectable H3 peptide deacetylation or nicotinamide formation,consistent with the previously described NAD-dependence (7, 8, 10). WhenNAD⁺ was included, robust deacetylation and nicotinamide formation wasobserved (FIG. 2). However, to our great surprise no significant amountsof ADP ribose were detected (FIG. 2). Nicotinamide and ADP ribose arethe predicted products from the hydrolysis of the NAD+glycosidic bond.To explore this finding and establish the degree of coupling betweenNAD⁺ consumption and AcLys-14H3 peptide deacetylation, the amount ofdeacetylated H3 formed and the amount of NAD⁺ consumed were determined(FIG. 3). Moles of product formed in the enzyme-catalyzed reaction werecalculated from standard curves generated with known amounts ofauthentic H3 peptide and nicotinamide. In reactions using severaldifferent initial concentrations of NAD⁺, every mole of NAD⁺ consumed bythe enzyme produced one mole of deacetylated H3 peptide, suggesting thatthese two chemical events are tightly coupled. Mass spectrometryconfirmed the identity of nicotinamide and deacetylated H3 peptide asthe HPLC product peaks co-eluting with authentic standards (FIGS. 1 and2). The previously described NAD⁺:nicotinamide exchange reaction wasconsistent with nicotinamide being a product of the reaction (7). Thetight coupling of nicotinamide formation and H3 deacetylation wasestablished further by comparing the steady-state rate of nicotinamideformation with the rate of AcLys-14H3 peptide deacetylation (FIG. 4).The NAD-concentration dependence of the steady-state rate of AcLys-14H3deacetylation matches exactly the rate of nicotinamide formation (FIG.4). Substrate saturation curves indicated that the K_(m) for NAD⁺ is 70μM and the K_(m) for AcLys-14H3 is estimated to be less than 500 nM. Thefact that NAD⁺ is consumed in the reaction indicates that it is not anallosteric regulator, but rather a bona fide substrate. Together, theseresults (FIGS. 2 and 3) suggested an exquisitely-linked enzymaticreaction in which NAD⁺ cleavage and deacetylation are directly coupled.

[0050] Next, we attempted to verify and quantify acetate as one of theobligate products. To accomplish this, H3 peptide was stoichiometricallyacetylated on Lys-14 by the histone acetyltransferase P/CAF using[³H]-AcCoA. Monoacetylated [³H]-AcLys-14H3 peptide was then purified byHPLC (FIG. 5A) and utilized as a substrate in the HST2 deacetylationreaction (FIG. 5B). To our surprise, the product profiles clearlyindicated that acetate was not the primary product in the reaction. TheHPLC elution position of the radioactive [3H]-acetate product (FIG. 5B)did not correspond to the position of authentic acetate (FIG. 1).Instead, the [³H]-labeled product eluted at a position that wassignificantly more hydrophobic than ADP-ribose. Upon completeconsumption of the [³H]-AcLys-14H3 peptide, only a trace amount ofacetate (<2% of total product formed) was detected in the reactions,whereas >98% of the original radioactivity from [³H]-AcLys-14H3 wastransferred to a novel acetate adduct (FIG. 5B). Control experiments inthe absence of enzyme did not result in transfer of acetate to this morehydrophobic form. Also, the acetate-adduct was not formed from anon-enzymatic solution reaction between the putative products[³H]-acetate, ADP ribose, nicotinamide, and H3 peptide.

[0051] To explore the idea that the acetate-adduct was a direct productof enzymatic deacetylation and not the product of a slowerside-reaction, a rapid-reaction single turnover experiment wasperformed. Using a quench-flow apparatus, HST2 was rapidly reacted withexcess NAD⁺ and sub-stoichiometric levels of [³H]-AcLys-14H3. Underthese conditions, the enzyme will perform only a single round ofcatalysis, allowing us to quantify the time-dependent loss of[³H]-AcLys-14H3 and the generation of the [³H]-acetate-adduct. Atvarious times between 30 ms and 8 s, the reactions were quenched andwere analyzed by HPLC and liquid scintillation counting. The progresscurves: (FIG. 5C) for the rapid single turnover clearly demonstratedthat AcLys-14H3 substrate consumption (rate constant of 2.0±0.1 s⁻¹) andacetate-adduct formation (rate constant of 2.3±0.2 s⁻¹) wereconcomitantly linked, providing strong evidence that the acetate-adductis a primary product of HST2-catalyzed deacetylation.

[0052] UV/VIS spectral analysis revealed that the acetate-adductabsorbed strongly at 260 nm, consistent with an analog containing anadenine ring. During catalytic turnover, the appearance of a newUV-absorbing species (FIG. 2) correlated exactly with the fractionscontaining the [³H]-labeled product (FIG. 5SB). Since ADP ribose was notdetected as a bona fide product of the reaction (FIG. 2), it was logicalto suggest that the novel product was an adduct between ADP ribose andacetate. In similar experiments with SIR2 and CobB, theidentical-eluting radioactive product was observed and, again, noacetate or ADP ribose were detected as primary products in thesereactions.

[0053] To confirm our hypothesis that an adduct between ADP ribose andacetate is the authentic product, HPLC fractions containing this productwere subjected to both MALDI and ESI mass spectral analysis for massdetermination. The novel adduct yielded a mass (positive molecular ionin MALDI) of 602 (FIG. 6A) consistent with the enzymatic formation ofacetyl-ADP ribose. For comparison, the mass results of an ADP-ribosestandard are displayed in FIG. 6B, indicating the predicted mass of 560.The difference of 42 between the two masses is equivalent to thedifference between replacing a hydrogen on an ADP-ribose hydroxyl withan acetyl group. Confirming these results, the identical mass was alsoobserved by separate ESI/MALDI analyses from samples submitted to twoindependent facilities. Consistent with the formation of an O-acetylbond to ribose, hydroxylamine treatment of the acetyl-ADP ribosecompound liberated free ADP ribose. Similarly, high glycineconcentrations also liberated ADP ribose from the HST2, SIR2, andCobB-catalyzed formation of acetyl-ADP ribose. Thus, we provide directevidence that the efficient histone/protein deacetylase reaction istightly coupled to the formation of a novel acetyl-ADP ribose product.One molecule of NAD⁺ and acetyl-lysine (histone) are readily catalyzedto one molecule of deacetylated lysine, nicotinamide and acetyl-ADPribose. Although mass spectrometry cannot provide direct information onthe position of the acetyl group on ADP ribose, the chemical evidencediscussed below suggests that the C₁ position of ribose is the initialsite of attachment in the reaction mechanism proposed in FIG. 7. InNAD⁺, C₁ forms the glycosidic bond to nicotinamide. FIG. 7 shows aproposed catalytic mechanism for the production of O-acetyl-ADP ribose.Acetyl-lysine condenses directly with the oxo-carbenium cation, whichresulted from the elimination of nicotinamide from NAD⁺. A hydroxide ionthen attacks this intermediate to form a tetravalent intermediate, whichcan collapse to produce 1-O-acetyl-ADP ribose (Compound I, FIG. 7). The1-O-acetyl-ADP ribose may then be attacked by the adjacent 2′ OH group(via enzyme-assisted general base catalysis) to produce the2-O-acetyl-ADP ribose (Compound II, FIG. 7). Once in bulk solution, the2-O-acetyl-ADP ribose appears to be in rapid equilibrium with itssolution migration adduct 3-O-acetyl-ADP ribose (Compound III, FIG. 7).Addition quantitative HPLC analysis followed by 2-dimentional NMR(heteronuclear multiple bond correlation) experiments have revealed themigration and equilibrium among the O-acetyl ADP ribose adducts depictedin FIG. 7. Searches of chemical data-bases for O-acetyl-ADP riboseproduced no matches, indicating that it is a novel compound produced bya novel enzymatic reaction.

[0054] While not wishing to be bound to any particular molecular theory,we propose the possible chemical mechanism for catalysis by the. SIR2family (as depicted in FIG. 7). It is well-established that manyNAD-dependent enzymes (NAD⁺ glycohydrolases, ribosyl transferases andADP-ribosyl cyclases) form a putative oxo-carbenium ADP-ribose cation asthe direct product of nicotinamide elimination (14-16). Given thisprecedent for oxo-carbenium cation formation and the previously observedNAD⁺:nicotinamide exchange reaction (7), SIR2 enzymes will likely form asimilar intermediate. Interestingly, in the case of the SIR2 enzymes,oxo-carbenium cation formation appears to require acetyl-lysine binding(and/or deacetylation). Only in the presence of acetyl-lysine and NAD⁺,can exogenously added nicotinamide exchange with the enzyme intermediateto reform NAD⁺ (7).

[0055] Given our findings, it appears that the previously reportedhistone/protein ADP-ribosyltransferase activity (9-11) is a lowefficiency side reaction that can be explained through the partialuncoupling of the intrinsic deacetylation/acetate ADP-ribosylationreactions. The fact that these enzymes are capable of anNAD⁺-nicotinamide exchange reaction suggests that the oxo-carbeniumcation of ADP ribose is at least partially susceptible to attack by thebase nucleophile. However, the proposed oxo-carbenium cation of SIR2enzymes appears to be exquisitely constructed to limit other possibleside-reactions, such as attack by H₂O or by nucleophilic amino-acid sidechains, which would result in ADP ribose or protein ADP-ribosylation,respectively. We observed, at most, protein ADP-ribosylation that is˜0.1% of the authentic reaction described in this study. Also, ADPribose was not detected as a primary enzymatic product. It may bepossible that some uncoupling of this reaction to yield proteinADP-ribosylation could result from perturbations in native proteinstructure (partially unfolded protein, mutagenesis, inappropriatereaction conditions), and from extremely high concentrations of analternate acceptor, such as reactive protein side-chains.

[0056] Why do the SIR2 enzymes go to great lengths to couplehistone/protein deacetylation to the formation of o-acetyl-ADP ribose?It is attractive to propose that this novel molecule has a uniquecellular function(s) that may be linked to SIR2's gene silencingeffects. This implicates O-acetyl-ADP ribose as an important signalingmolecule in biochemical pathways in which other enzymes/proteins mayutilize O-acetyl-ADP ribose to elicit the proper cellular response. Suchtargets might include ATP-dependent chromatin remodeling enzymes,histone/protein acetyltransferases or poly(ADP-ribosyl)transferases. Itis interesting to note that poly(ADP-ribosyl)transferases utilize NAD⁺to poly(ADP-ribosyl)ate proteins involved in the metabolism of nucleicacids and in the maintenance of chromatin architecture (15, 17). Oneintriguing possibility is that O-acetyl-ADP ribose could bindpoly(ADP-ribosyl)transferases and block poly(ADP-ribosyl)ation.Moreover, NAD⁺ levels in cells is inversely affected by the level ofprotein poly(ADP-ribosyl)ation (15, 17). Sincepoly(ADP-ribosyl)transferases and SIR2 enzymes exhibit similar K_(m)values (˜50-70 μM) for NAD⁺, they may compete for the available NAD⁺ andoppose each another's function. Recently, the observation that caloricrestriction leading to increased life-span appears to be linked throughan NAD⁺ and SIR2-dependent pathway (18), raises the possibility thatO-acetyl-ADP ribose may play a role in this phenomenon. It is importantto note that bacteria do not have histones and yet they do haveSIR2-like proteins with similar activity as has been shown here forHST2/SIR2/CobB/hSIRT2. Thus, histones need not be the only substratesfor deacetylation by these enzymes. Identification of the authenticproducts and the catalytic mechanism of the SIR2-like enzymes hasprovided the initial basis for understanding the cellular role(s) playedby this important family of proteins.

EXAMPLE II

[0057] To provide evidence for the existence of human cellularproteins/enzymes that are capable of binding and catalyzing theconversion or decomposition of O-acetyl-ADP ribose, human HeLa cellextracts were generated and the ability of proteins within the extractto catalyze the decomposition of O-acetyl-ADP ribose was assessed.Radio-labeled O-acetyl-ADP ribose was generated and purified asdescribed above, and was added to the HeLa cell extract. As a control,O-acetyl-ADP ribose was added to a buffer control. At time points up to3 hrs, the samples were analyzed for the amount of O-acetyl-ADP riboseremaining (by HPLC as described above). The results, shown in FIGS. 8Aand 8B clearly demonstrate the ability of the human cell extract toconvert O-acetyl-ADP ribose to a distinct, but as yet unidentifiedspecies. These findings suggest that O-acetyl-ADP ribose could be actingas a second messenger, where additional proteins/enzymes are capable ofterminating the signal through the enzymatic decomposition ofO-acetyl-ADP ribose. These data also provide evidence for the existenceof proteins/enzymes that bind/utilize O-acetyl-ADP ribose.

[0058] Enzymatic Generation of Novel O-Acetyl-Nucleotide Analogs

[0059] We have demonstrated that different nucleotide analogs, such asNicotinamide Guanine Dinucleotide (NGD), can serve as substrates for theSIR2 family of enzymes, generating the novel O-acetyl-NDP riboseproduct. The details of the reaction conditions are as described in thematerials and methods section. This enzymatic process may be used toadvantage to generate novel O-acetyl-nucleotide analogs. These analogsmay be used in the development of inhibitors for SIR2 and SIR homologs,or for the proteins/enzymes that normally bind/utilize O-acetyl-ADPribose. More importantly, these enzyme-derived nucleotide analogs mayserve as potent therapeutics for cancer and aging, releasing the blockfor transcriptionally-silenced genes that have been inappropriatelysilenced during tumorigenesis and aging, as discussed earlier.

[0060] Methods for Enzymatic Generation of Novel O-acetyl-NucleotideAnalogs

[0061] Typical HST2 steady-state reactions to monitor deacetylation ofAcLys-14H3 peptide or nicotinamide formation with nicotinamide guaninedinucleotide (NGD) as an alternative substrate to NAD were carried outat various concentrations of AcLys-14H3 peptide (5-100 μM) and NGD(10-400 μM). During the linear portion of the initial velocity, thereactions were monitored by reverse phase HPLC analysis on a BeckmanUltrasphere column (4.6 mm×15 cm). Samples were injected in 0.05%trifluoroacetic acid (TFA)/H₂O for 1 min before a linear gradient of0-40% acetontrile in 1-41 min.

EXAMPLE III

[0062] The data presented in the previous examples indicates thatO-acetyl ADP ribose is a novel molecule demonstrating unique cellularfunction(s) that may be linked to SIR2's gene silencing or otherphysiological effects. It appears that O-acetyl-ADP ribose has animportant signaling role in which other enzymes/proteins utilizeO-acetyl-ADP ribose to elicit the proper cellular response. Such targetsmight include ATP-dependent chromatin remodeling enzymes,histone/protein acetyltransferases or poly(ADP-ribosyl)transferases.Also, the observation that caloric restriction leading to increasedlife-span appears to be linked through an NAD⁺ and SIR2-dependentpathway, raises the possibility that O-acetyl-ADP ribose may play a rolein this phenomenon.

[0063] To explore the function of SIR2-like enzymes and of O-acetyl-ADPribose, microinjection analyses in living cells were performed whichhave provided evidence for phenotypic effects caused by SIR2- orO-acetyl-ADP ribose. The evidence presented herein suggests thatcellular enzymes that metabolize/convert O-acetyl-ADP ribose areimportant in maintaining cell cycle control. Micro-injection assays wereperformed in starfish and sea urchin ooctyes. Historically echinodermdevelopment has been a biochemically tractable and well-defined systemto assess biological function of proteins, bio-active compounds and fordetailed analyses of the cell cycle. This system is attractive for itsamenability to quantitative microinjection assays, cell cycle analyses,biochemical fractionation and microscopic imaging of eggs and earlyembryos. Initially, we hypothesized that if O-acetyl-ADP ribose producedby SIR2 was involved in SIR2's gene silencing ability, thenmicro-injection of O-acetyl-ADP ribose into a maturing oocyte shouldgive rise to a phenotypic effect on the developing animal, perhapsthrough perturbations in normal gene expression patterns. Immatureoocytes were micro-injected with HPLC-purified O-acetyl-ADP ribose orwith a buffer control. The oocytes were allowed to mature by theaddition of 1-methyladenine and assessed for developmental alterations.

[0064] Compared to control injections, oocytes injected witho-acetyl-ADP ribose exhibited a delay in germinal vesicle breakdown(GVBD), or a complete block in oocyte maturation as assessed by GVBD andpolar body extrusion. See FIG. 9 and Table I below. The dose-dependencyindicated that below 0.32 mM no effect was observed. Given the amountsof O-acetyl-ADP ribose required for these phenotypes, we suspected thatthe injected O-acetyl-ADP ribose might be metabolized over time,resulting in a loss of the compound during the 24 hours of the assay.Supporting this assertion, we have demonstrated that HeLa cell extractsare capable of consuming O-acetyl ADP ribose, relative to a buffercontrol. See Example II. Together these data suggest the existence ofenzymes that metabolize O-acetyl ADP ribose.

[0065] To complement these studies, we micro-injected active yeast HST2and human SIR2 enzymes instead of O-acetyl-ADP ribose. We hypothesizedthat if acetylated protein substrates were available in the oocyte, thenmicro-injected enzyme would provide a more constant source ofO-acetyl-ADP ribose. As a control, a catalytically impaired mutant ofHST2 (R45A) was employed to ensure that any effects were due to thecatalytic activity of the enzyme. The R45A mutant is >50-fold lower incatalytic efficiency (k_(cat) and k_(cat)/K_(m) for acetyl-peptide)compared to wild type enzyme (unpublished data). TABLE I Effects ofO-acetyl ADP ribose on starfish oocyte maturation. Injected compound 1-2hr compete immediate conc. in cytoplasm no effect delay block death O-AcADP ribose 10 mg/ml (16 mM) 2/2 3.0 mg/ml (5 mM) 2/3 1/3 1.7 mg/ml (3mM) 6/6 1.0 mg/ml (1.6 mM) 1/23 22/23 0.5 mg/ml (0.8 mM) 10/16  6/16 0.2mg/ml (0.32 mM) 7/8 1/8 0.1 mg/ml (0.16 mM) 16/16 0.01 mg/ml (0.016 mM)8/8

[0066]Asterina miniata immature oocytes were microinjected with theamounts of O-acetyl ADP ribose in PBS. The maturation hormone1-methyladenine was added (10 μM) 25-30 minutes post-injection. Oocyteswere monitored for GVBD and polar body extrusion. Non-injected andcontrol injected oocytes showed synchronous GVBD within 35-45 minutes(depending on the batch). The number of oocytes per treatment is given.Combined results of 3 separate experiments (3 batches of O-acetyl ADPribose).

[0067] First, effects of HST2 (wild type and mutant) and human SIRT2enzymes on sea star oocyte maturation were assessed. See Table II below.Asterina miniata immature oocytes were microinjected with the givenenzymes in PBS. The maturation hormone 1-methyladenine was added (10 μM)25-30 minutes post-injection. Oocytes were monitored for GVBD and polarbody extrusion. Non-injected and control injected oocytes showedsynchronous GVBD within 35-45 minutes (depending on the batch). A dosageresponse analysis was performed using final enzyme concentrations thatranged between 25-200 nM. Above 72 nM, both hSIRT2 and HST2 caused acomplete block in maturation (no GVBD or polar body extrusion). At 60nM, both enzymes caused a 2 hr delay in GVBD and polar body extrusion.With 25 nM hSIRT2 or HST2, no effect was observed. The catalytic mutantR45A displayed no observed effects at any concentration from 25-200 rM.Representative oocyte images are shown in the FIGS. 10A-10C. These dataindicate a clear dosage dependence with the active enzymes, and suggestthat hSIRT2 and HST2 are involved in a highly specific block in oocytematuration. TABLE II Effects of yeast HST2 and human SIRT2 enzymes onsea star oocyte maturation. Enzyme Final conc (nM) n Effects/Notes HST225 10 no observed effect (2 preps) 60 5 2 hr delay in GVBD 72 22complete block 200 10 complete block in 9/10; 3 hr delay 1/10 HST2 R45A25 5 no observed effect 60 5 no observed effect 72 13 no observed effect200 5 no observed effect hSIRT2 25 5 no observed effect (1 prep) 60 5 2hr delay in GVBD and polar body extrusion 200 5 complete block # Thenumber of oocytes per treatment (n) is given. Data are from fourseparate batches of oocytes. All oocytes for a given treatment respondedin the same way unless indicated otherwise (e.g. see 200 nM HST2injection).

[0068]Asterina miniata immature oocytes were microinjected with thegiven enzymes in PBS. The maturation hormone 1-methyladenine was added(10 μM) 25-30 minutes post-injection. Oocytes were monitored for GVBDand polar body extrusion. Non-injected and control injected oocytesshowed synchronous GVBD within 35-45 minutes (depending on the batch).The number of oocytes per treatment (n) is given. Data are from fourseparate batches of oocytes. All oocytes for a given treatment respondedin the same way unless indicated otherwise (e.g. see 200 nM HST2injection).

[0069] To examine whether these SIR2 enzymes caused any phenotypiceffects on early embryonic cell division, HST2 or the R45A mutant weremicro-injected into one cell of the two-cell stage embryos. Asterinaminiata oocytes were matured by addition of the hormone 1-methyladenineand then fertilized. One cell zygotes were then placed in injectionchambers. After the first cell division, one daughter blastomere wasmicroinjected with the indicated enzyme (see table and figure below).Embryos were monitored for further cell divisions over time. Thenon-injected daughter blastomere served as the internal control in eachexperiment. At and above 72 nM, HST2 caused a complete block in celldivision either at the next or subsequent division, while thenon-injected daughter blastomere developed normally. Additionally, cellsthat did make it through the next division displayed a slight delaybefore a complete block in the next round. Most cells injected at thelower 25 nM HST2 developed normally. The R45A mutant exhibited nophenotype at any concentration. Again, there was a clear dosage effectthat paralleled the effect observed on oocyte maturation. Given the lowconcentrations necessary for the blocks, the dosage effects, and therequirement for active enzyme, collectively suggest that these SIR2enzymes are causing a specific and highly potent cell cycle block. TABLEIII Effects of HST2 on early embryonic cell division. Final concEffects: 3^(rd) (nM) in Effects: Next cell Effects: Enzyme blastomere ncell division division other HST2 25 5 4/5 normal Normal Normal (2preps) development 1/5 as slight Normal Normal delay development HST2 (272 10 4/10 slight Complete — preps) delay block 6/10 no — division(complete block) HST2 200 7 2/7 slight Complete — (2 preps) delay block5/7 no — division (complete block) HST2 R45A 25 5 Normal Normal Normaldevelopment HST2 R45A 72 3 Normal Normal Normal development HST2 R45A200 5 normal Normal Normal development # The non-injected daughterblastomere served as the internal control in each experiment. The numberof injected embryos per treatment is given (n). Data are from twoseparate batches of oocytes.

[0070]Asterina miniata oocytes were matured by addition of the hormone1-methyladenine and then fertilized. One cell zygotes were then placedin injection chambers. After the first cell division, one daughterblastomere was microinjected with the indicated enzyme. Embryos weremonitored for further cell divisions over time. The non-injecteddaughter blastomere served as the internal control in each experiment.The number of injected embryos per treatment is given (n). Data are fromtwo separate batches of oocytes.

[0071] Quite strikingly, injection of pure O-acetyl ADP ribose mimickedthe oocyte maturation block that was observed when low nM levels ofactive enzyme were injected. Not only did O-acetyl ADP ribose blockoocyte maturation, but it also delayed apoptosis that normally occursafter ˜24 hrs if the oocyte is not fertilized. Both SIR2 enzymes andO-acetyl ADP ribose appear to have an effect on oocyte longevity in thiscontext. The results provide additional biological/biochemical data thatindicate the product O-acetyl ADP ribose plays an integral, if notsufficient role in SIR2-dependent phenotypic alterations. The anti-agingphenotypes and the cell-cycle blot suggest the importance of our initialdiscovery (unique reaction of SIR2-family enzymes and the production ofO-acetyl ADP ribose) further supports the ramifications of O-acetyl ADPribose and it's analogs as potential cancer therapeutics or anti-agingdrugs.

REFERENCES

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[0089] While certain of the preferred embodiments of the presentinvention have been described and specifically exemplified above, it isnot intended that the invention be limited to such embodiments. Variousmodifications may be made thereto without departing from the scope andspirit of the present invention, as set forth in the following claims.

What is claimed is:
 1. A histone deacetylase-reaction product,O-acetyl-ADP ribose.
 2. A pharmaceutical composition comprising thereaction product of claim 1 in a biologically acceptable carrier medium.3. An antibody immunologically specific for O-acetyl ADP ribose.
 4. Theantibody of claim 3, wherein said antibody is monoclonal.
 5. Theantibody of claim 3, wherein said antibody is polyclonal.
 6. A methodfor isolating test compounds having binding affinity for O-acetyl ADPribose comprising: a) providing immobilized O-acetyl ADP ribose; b)contacting said immobilized O-acetyl ADP ribose with a test compoundsuspected of having binding affinity for said O-acetyl ADP ribose; andc) eluting said immobilized O-acetyl ADP ribose and assessing eluate forthe presence of said test compound.
 7. The method as claimed in claim 6,wherein said test compound is a protein present in a cell extract. 8.The method as claimed in claim 6, wherein said O-acetyl ADP ribose isimmobilized on a column.
 9. The method as claimed in claim 6, whereinsaid O-acetyl ADP ribose is immobilized on a multiwell plate.
 10. Amethod for producing O-acetyl ADP ribose in a cell, comprising: a)transforming a host cell with a gene selected from group consisting ofHST2, SIR2, HST1, HST3, HST4, CobB and homologues thereof; b) isolatinga protein encoded by said gene from said transformed host cell; c)contacting said isolated protein with detectably labeled AcLYs-14H3peptide and NAD+ under conditions suitable for deacetylation to occur;and d) isolating said O-acetyl ADP ribose.
 11. O-acetyl ADP riboseisolated by the method of claim
 10. 12. A method as claimed in claim 10,wherein said detectable label is selected from the group consisting offluorescein, rhodamine, radioactive isotopes, and chemiluminescencelabels.
 13. An nucleotide analog of O-acetyl ADP ribose selected fromthe group consisting of O-acetyl purine nucleotide ribose and O-acetylpyrimidine nucleotide ribose.
 14. The nucleotide analog of claim 13,wherein said analog is non-hydrolyzable.
 15. The nucleotide analog ofclaim 13, further comprising a detectable label.
 16. The nucleotideanalog of claim 15, wherein said detectable label is selected from thegroup consisting of fluorescein, rhodamine, radioactive isotopes, andchemiluminescence labels.
 17. O-acetyl-ADP ribose as claimed in claim 1,wherein said O-acetyl-ADP ribose is detectably labeled.
 18. O-acetyl-ADPribose as claimed in claim 17, wherein said detectable label is selectedfrom the group consisting of fluorescein, rhodamine, radioactiveisotopes, and chemiluminescence labels.